WO2008134123A1 - Ul97 for treatment of protein aggregate disorders - Google Patents

Abstract

Compositions and methods are described for treating or preventing protein aggregate disorders comprising administering a viral kinase or fragments, variants or isoforms thereof to a subject. cotransfected

Description

Attorney Docket No. 20674-066 WOl

UL97 for Treatment of Protein Aggregate Disorders

CROSS-REFERENCE TO PRIORITY APPLICATIONS

This application claims priority to U.S. Provisional Application No. 60/914,911, filed April 30, 2007, which is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant Nos. NOI-AI- 30049 and NS051422 awarded by the National Institutes of Health. The government has certain rights in the invention. BACKGROUND

Abnormalities of protein folding, oligomerization, aggregation or deposition play an important role in the pathophysiology of a diverse set of chronically progressive degenerative disorders. For example, aggresomes are highly dynamic cellular structures that act to sequester inappropriately aggregated proteins, and their formation is linked to pathogenic processes in aggregative diseases such as

Alzheimer's, Parkinson's and Huntington's diseases. Many prion and viral proteins are also targeted to these structures. To date, therapeutic agents and completely effective treatments for diseases characterized by the presence of inclusions, aggresomes and plaques are not available. SUMMARY

Provided are compositions and methods for treating protein aggregate disorders in a subject comprising administering to the subject a viral kinase. Preferably, the viral kinase is a UL97 or a homolog of UL97 or fragments, variants or isoforms thereof. The details of one or more embodiments are set forth in the accompanying drawings and description below. Other features, objects, and advantages will be apparent from the description and drawings, and from the claims.

DESCRIPTION OF DRAWINGS Figure 1 shows IEl and pp71 promote the formation of pp65-GFP nuclear aggregates. Cells co-expressing the pp65-GFP fusion protein with the other viral Attorney Docket No. 20674-066WO1

proteins shown were counted and the percentage containing nuclear aggregates was calculated. The value shown is the average of 10 fields with the standard deviation represented by the error bars.

Figures 2 A-U show inhibition of aggregate formation by UL97 kinase. Aggregates induced through the expression of pp65, pp71 and GFP-GCP 170* are shown in panels A-C, respectively. Aggregate formation is inhibited in cells that co- express pp65-GFP and UL97 (panels D-F). The kinase negative mutant (K355M) is unable to inhibit aggregation of pp65-GFP and is recruited to the aggregates (panels G-I). The kinase also inhibits the formation of pp71 aggregates in co-transfected cells (panels J-L). The K355M mutant is unable to affect pp71 aggregates and is recruited to the aggregates (panels M-O). Aggresomes induced through the expression of the cellular protein, GFP-GCP170* are inhibited by the kinase (panels P-R). Aggregation of GFP-GCP 170* is unaffected by the K355M mutant, which is specifically recruited to cytoplasmic aggresomes (panels S-U). Figures 3 A-F show that UL97 kinase reduces the number of PML domains in

COS7 cells. Cells were transfected with (A) ppUL44, (B) IEl, (C) pp71, (D) UL97, (E) K355M, and (F) UL97 with the addition of MBV. Expression of pUL97 reduced the number of PML bodies in a kinase dependent manner. Values shown in each panel are average number of PML domains per cell and reflect the average of at least 50 cells with the standard error of the mean shown.

Figure 4 shows that UL97 kinase activity is required for the hyperphosphorylation of Rb in infected cells. Cell lysates from mock infected cells, cells infected with AD 169, or cells infected with a UL97 deleted virus (UL97Δ) were separated on polyacrylamide gels and transferred to PVDF membranes. Samples were obtained at 24 and 72 hours following infection and half of the infections occurred in the presence of MBV, as indicated in the figure. Shown are immunoblots using monoclonal antibodies to the proteins shown to the left of the figure. Hyperphosphorylated forms of the retinoblastoma protein (Rb) are reduced when the kinase is deleted or when its activity is inhibited with maribavir (MBV). Figure 5 A shows Rb binding motifs in pUL97. Amino acid sequences of viral proteins containing LxCxE (SEQ ID NO:1) and LxCxD (SEQ ID NO:2) motifs were aligned with the motifs identified in pUL97. Sequences for HCMV LxCxE Attorney Docket No. 20674-066WO1

(NP_040032.1) (SEQ ID NO:3), CCMV UL97 (NP_612729) (SEQ ID NO:4), SV40 large T (NP 043127) (SEQ ID NO:5), HAdV ElA (ABK35030.1) (SEQ ID NO:6), HPV 16 E7, (AAD33253.1) (SEQ ID NO:7), HHV-6AU69 (NP_042962.1) (SEQ ID NO:8), HHV-6B U69 (T44214) (SEQ ID NO:9), HHV-7 U69 (YP 073809.1) (SEQ ID NO: 10), HCMV pp71 (NP 040017) (SEQ ID NO: 11), and HCMV LxCxD

(NP_040032.1) (SEQ ID NO: 12), are shown with the consensus sequence (SEQ ID NO:20).

Figure 5B shows epitope tagged versions of pUL97 expressed in COS7 cells and immunoprecipitated with a monoclonal antibody to the V5 epitope tag. The K355M mutation eliminates kinase activity, whereas C428G and C141G disrupt the

LxCxD (SEQ ID NO:2) and LxCxE (SEQ ID NO:1) motifs, respectively. UL89 and ULl 04 fusion proteins serve as negative controls and Rb alone was used as a positive control. Immunoblot shown in the top panel shows the expression of the immunoprecipitated fusion proteins. The bottom blot shows the Rb bound to the precipitated UL97 proteins but not to the negative controls.

Figure 6 shows the LxCxE (SEQ ID NO: 1) motif in the kinase is required for the inhibition of aggregate formation. Cells co-expressing pp65-GFP with the proteins shown were counted and the percentage of cells that exhibited nuclear aggregates were calculated. The values shown are the average of three separate experiments with the standard deviations shown.

Figure 7 shows a schematic model of UL97 kinase function in aggregate formation.

Figure 8 shows HHV-6B U69 protein kinase is inhibited by MBV. Cells were transfected with constructs expressing UL27, UL97, UL69 or UL69B K219M and treated with 15 μM MBV (dark gray bars) or untreated (light gray bars). Aggregation of pp65-GFP is inhibited by both the HCMV UL97 kinase and the HHV-6 U69 kinase (light gray bars). Inhibition of aggregation is kinase dependent since the U69B K219M mutation, which eliminates an essential lysine, is incapable of inhibiting aggregation. Treatment of infected cells with 15 μM MBV reduces the ability of UL97 and U69 to inhibit aggregation (dark gray bars).

Figures 9A, 9B and 9C show that UL97 prevented aggregation of GFP170*. COS7 cells were transfected with GFP170* alone (Fig. 9A), with GFP170* and V5- Attorney Docket No. 20674-066WO1

tagged wild-type UL97 (Fig. 9B) or with GFP 170* and V5-tagged inactive UL97/K355M (Fig. 9C). After 24 hows, cells were processed for immunofluorescence with anti-V5 antibodies. GFP 170* formed large cytoplasmic and nuclear aggregates when expressed alone (Fig. 9A). Aggregates were also detected when GFP170* was co-expressed with the inactive UL97/K355M (Fig. 9C).

Aggregates were not detected in cells that co-expressed GFP 170* and active UL97 (Fig. 9B).

Figures 1OA, 1OB and 1OC show UL97 prevented aggregation of Httexl-82Q- YFP. HeLa cells were transfected with HttExonl-82Q-YFP alone (Fig. 10A), with HttExonl-82Q-YFP and V5-tagged wild-type UL97 (Fig. 10B) or with HttExonl-

82Q-YFP and V5-tagged inactive UL97/K355M (Fig. 10C). After 48 hours, cells were processed for immuno-fluorescence with anti-V5 antibodies. In the absence of UL97, HttExonl-82Q-YFP formed large cytoplasmic and nuclear aggregates (Fig. 10A). Inactive UL97/K355M did not prevent aggregation (Fig. 10C). Aggregates were not detected or were greatly reduced in cells that also expressed UL97 (Fig.

10B).

Figures HA, HB and IIC show UL97 prevented aggregation of AT3-72Q. HeLa cells were transfected with myc-tagged AT3-72Q alone (Fig. 11A), with myc- tagged AT3-72Q and V5-tagged wild-type UL97 (Fig. 1 IB) or with myc-tagged AT3- 72Q and V5-tagged inactive UL97/K355M (Fig. HC). After 72 hours, cells were processed for immunofluorescence with anti-myc and anti-V5 antibodies. When expressed alone, AT3-72Q formed large cytoplasmic and nuclear aggregates (Fig. HA). Inactive UL97/K355M did not prevent cytoplasmic or nuclear aggregation (Fig. 11C). Large aggregates were not detected in cells that expressed AT3-72Q and UL97 (Fig. HB).

Figure 12 is a graph showing quantitation of UL97 prevention of aggregation of AT3-72Q. HeLa cells were transfected with myc-tagged AT3-72Q alone, with myc- tagged AT3-72Q and V5-tagged wild-type UL97 or with myc-tagged AT3-72Q and V5-tagged inactive UL97/K355M. After 72 hours, cells were processed for immunofluorescence with anti-myc and anti-V5 antibodies. Cells containing both proteins were scored for the presence and size of aggregates. Attorney Docket No. 20674-066WO1

DETAILED DESCRIPTION

Many degenerative diseases are associated with the oligomerization, aggregation or fibrillization of various proteins. Provided herein are methods and compositions that are useful in the prevention, treatment or modulation of protein aggregate diseases. Specifically, a method of treating or preventing a protein aggregate disease or disorder in a subject is provided that includes administering a UL97 or a UL97 homolog or a fragment or variant thereof to the subject. I. Viral Kinases

Each of the human herpesviruses (HHV) encode at least one well-conserved serine/threonine protein kinase that are important in viral infection. Herpes simplex virus (HSV) ULl 3 and Epstein-Barr virus (EBV) BGLF4 phosphorylate eukaryotic elongation factor 1 delta , and HSV UL 13 and human cytomegalovirus (HCMV) pUL97 both phosphorylate the carboxyl-terminal domain of RNA polymerase II. Many other activities on cellular proteins have been described, such as the activation of cdc2 by HSV UL13, the inhibition of histone acetylation and activation of protein kinase A by HSV UL3.

The UL97 protein kinase is a tegument protein expressed with early/late kinetics, which autophosphorylates amino terminal serine and threonines. A recombinant virus with a large deletion in UL97 replicates poorly in its absence and virus titers are reduced more than 100-fold showing that it is critical in the replication of the virus. Virion morphogenesis in the nucleus is also impaired, resulting in the inappropriate aggregation of pp65 and the sequestration of viral proteins in large nuclear aggregates. As described herein, UL97 inhibits the formation of aggresomes and PML bodies in a kinase dependent manner, and its activity is required for the inactivation of Rb.

Provided for use in the methods and compositions herein are a UL97, a UL97 homolog, a UL97 fragment,, a UL97 homolog fragment, variants or iso forms of a UL97 or a UL97 homolog. As used herein, a UL97 refers to UL97 kinase from cytomegalovirus (CMV) and homologs, variants and isoforms thereof. Homologs of UL97 kinase include HSV UL13, EBV BGLF4, HHV-6A UL69, HHV-6B UL69 and

HHV-7 UL69. There are a variety of sequences that are disclosed on Genbank, at www.pubmed.gov and these sequences and others are herein incorporated by Attorney Docket No. 20674-066WO1

reference in their entireties as well as for individual subsequences contained therein. For example, the amino acid and nucleic acid sequences of human CMV UL97 can be found at GenBank Accession Nos. NP_040032.1 and NC OO 1347.2, respectively. The amino acid and nucleic acid sequences of chimpanzee CMV can be found at GenBank Accession Nos. NP 612729 and NC 003521.1, respectively. The amino acid and nucleic acid sequences of HHV-6A U69 can be found at GenBank Accession Nos. NP_042962.1 and NC OO 1664.1, respectively. The amino acid sequence of HHV-6B U69 can be found at GenBank Accession Nos. T44214. The amino acid and nucleic acid sequences of HHV-7 U69 can be found at GenBank Accession Nos. YP 073809.1 and NC 001716.2, respectively. Thus provided are amino acid sequences of the viral kinases comprising an amino acid sequence at least about 70%, 75%, 80%, 85%, 86%, 90%, 95%, 98%, 99% or more identical to the sequence found at the aforementioned GenBank accession numbers. Also provided are nucleic acids encoding viral kinases comprising a nucleotide sequence at least about 70%, 75%, 80%, 85%, 86%, 90%, 95%, 98%, 99% or more identical to the nucleotide sequence found at the aforementioned GenBank accession numbers or complement thereof. As used herein, the term peptide, polypeptide, protein or peptide portion is used broadly herein to mean two or more amino acids linked by a peptide bond. Protein, peptide and polypeptide are also used herein interchangeably to refer to amino acid sequences. The term fragment is used herein to refer to a portion of a full- length polypeptide or protein. It should be recognized that the term polypeptide is not used herein to suggest a particular size or number of amino acids comprising the molecule and that a peptide of the invention can contain up to several amino acid residues or more. As with all peptides, polypeptides, and proteins, it is understood that substitutions in the amino acid sequence of the UL97, UL97 homolog or fragments of UL97 or UL97 homolog can occur that do not alter the nature or function of the peptides, polypeptides, or proteins. Such substitutions include conservative amino acid substitutions and are discussed in greater detail below. The polypeptides provided herein have a desired function. The polypeptides as described herein selectively bind retinoblastoma (Rb), pl30 or plO7. By binding is meant a detectable binding at least about 1.5 times the background of the assay Attorney Docket No. 20674-066WO1

method. For selective or specific binding such a detectable binding can be detected for a given agent but not a control antigen or agent. The polypeptide provided herein inactivates Rb, pi 30 or pi 07. The polypeptide can cause hyperphosphorylation of Rb. The polypeptides are tested for their desired activity using the in vitro assays described herein, or by analogous methods, after which their therapeutic, diagnostic or other purification activities are tested according to known testing methods.

The polypeptides described herein can be modified and varied so long as the desired function is maintained. It is understood that one way to define any known variants and derivatives or those that might arise, of the disclosed genes and proteins herein is through defining the variants and derivatives in terms of homology to specific known sequences. Specifically disclosed are variants of a UL97 or a UL97 homolog and nucleic acids encoding a UL97 or a UL97 homolog herein disclosed which have at least, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 percent homology to the stated sequence. Those of skill in the art readily understand how to determine the homology of two proteins or nucleic acids, such as genes. For example, the homology can be calculated after aligning the two sequences so that the homology is at its highest level.

In general, it is understood that one way to define any known variants and derivatives or those that might arise, of the disclosed genes and proteins herein, is through defining the variants and derivatives in terms of homology to specific known sequences. This identity of particular sequences disclosed herein is also discussed elsewhere herein. In general, variants of genes and proteins herein disclosed typically have at least, about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent homology to the stated sequence or the native sequence. Those of skill in the art readily understand how to determine the homology of two proteins or nucleic acids, such as genes. For example, the homology can be calculated after aligning the two sequences so that the homology is at its highest level.

Another way of calculating homology can be performed by published algorithms. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman Adv. Appl. Math. 2: 482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. MoL Biol. 48: 443 Attorney Docket No. 20674-066 WOl

(1970), by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by inspection. The same types of homology can be obtained for nucleic acids by for example the algorithms disclosed in Zuker, M. Science 244:48-52, 1989, Jaeger et al. Proc. Natl. Acad. Sci. USA 86:7706-7710, 1989, Jaeger et al. Methods Enzymol. 183:281- 306, 1989 which are herein incorporated by reference for at least material related to nucleic acid alignment. It is understood that any of the methods typically can be used and that in certain instances the results of these various methods may differ, but the skilled artisan understands if identity is found with at least one of these methods, the sequences would be said to have the stated identity, and be disclosed herein.

Fragments, variants, or isoforms of a UL97 or a UL97 homolog are provided. It is understood that these terms include functional fragments and functional variants. For example, fragments can include any portion of the UL97 or UL97 homolog.

Fragments optionally include an LxCxE (SEQ ID NO: 1) motif, a DSSE (SEQ ID NO: 13) motif, and/or an LxCxD (SEQ ID NO:2) motif.

The variants are produced by making amino acid substitutions, deletions, and insertions, as well as post-translational modifications. Variations in post-translational modifications can include variations in the type or amount of carbohydrate moieties of the protein core or any fragment or derivative thereof. Variations in amino acid sequence may arise naturally as allelic variations (e.g., due to genetic polymorphism) or may be produced by human intervention (e.g., by mutagenesis of cloned DNA sequences), such as induced point, deletion, insertion and substitution mutants. These modifications can result in changes in the amino acid sequence, provide silent mutations, modify a restriction site, or provide other specific mutations. Protein variants and derivatives can involve amino acid sequence modifications. For example, amino acid sequence modifications typically fall into one or more of three classes: substitutional, insertional or deletional variants. Insertions include amino and/or carboxyl terminal fusions as well as intrasequence insertions of single or multiple amino acid residues. Insertions ordinarily will be smaller insertions than those of amino or carboxyl terminal fusions, for example, on Attorney Docket No. 20674-066 WOl

the order of one to four residues. Deletions are characterized by the removal of one or more amino acid residues from the protein sequence. Typically, no more than about from 2 to 6 residues are deleted at any one site within the protein molecule. These variants ordinarily are prepared by site specific mutagenesis of nucleotides in the DNA encoding the protein, thereby producing DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture. Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, for example Ml 3 primer mutagenesis and PCR mutagenesis. Amino acid substitutions are typically of single residues, but can occur at a number of different locations at once; insertions usually will be on the order of about from 1 to

10 amino acid residues; and deletions will range about from 1 to 30 residues. Deletions or insertions preferably are made in adjacent pairs, i.e. a deletion of 2 residues or insertion of 2 residues. Substitutions, deletions, insertions or any combination thereof may be combined to arrive at a final construct. The mutations must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary mRNA structure. Substitutional variants are those in which at least one residue has been removed and a different residue inserted in its place. Such substitutions generally are made in accordance with the following Table 1 and are referred to as conservative substitutions. TABLE 1 : Amino Acid Substitutions

Ammo Acid Substitutions (others are known in the art)

Ala Ser, GIy, Cys

Arg Lys, GIn, Met, He

Asn GIn, His, GIu, Asp

Asp GIu, Asn, GIn

Cys Ser, Met, Thr

GIn Asn, Lys, GIu, Asp

GIu Asp, Asn, GIn

GIy Pro, Ala

His Asn, GIn

He Leu, VaI, Met Attorney Docket No. 20674-066 WOl

Leu He, VaI, Met

Lys Arg, GIn, Met, He

Met Leu, He, VaI

Phe Met, Leu, Tyr, Tip, His

Ser Thr, Met, Cys

Thr Ser, Met, VaI

Tip Tyr, Phe

Tyr Trp, Phe, His

VaI He, Leu, Met

Substantial changes in function or immunological identity are made by selecting substitutions that are less conservative than those in Table 1, i.e., selecting residues that differ more significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site or (c) the bulk of the side chain. The substitutions which in general are expected to produce the greatest changes in the protein properties will be those in which (a) a hydrophilic residue, e.g. seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g. leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, e.g., lysyl, arginyl, or histidyl, is substituted for (or by) an electronegative residue, e.g., glutamyl or aspartyl; or (d) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having a side chain, e.g., glycine, in this case, (e) by increasing the number of sites for sulfation and/or glycosylation.

As used herein, modification with reference to a polynucleotide or polypeptide, refers to a naturally-occurring, synthetic, recombinant, or chemical change or difference to the primary, secondary, or tertiary structure of a polynucleotide or polypeptide, as compared to a reference polynucleotide or polypeptide, respectively (e.g., as compared to a wild-type polynucleotide or polypeptide). Modifications include such changes as, for example, deletions, insertions, or substitutions. Polynucleotides and polypeptides having such mutations can be isolated or generated using methods well known in the art. Attorney Docket No. 20674-066 WOl

Nucleic acids that encode the aforementioned peptide sequences, variants and fragments thereof are also disclosed. These sequences include all degenerate sequences related to a specific protein sequence, i.e. all nucleic acids having a sequence that encodes one particular protein sequence as well as all nucleic acids, including degenerate nucleic acids, encoding the disclosed variants and derivatives of the protein sequences. Thus, while each particular nucleic acid sequence may not be written out herein, it is understood that each and every sequence is in fact disclosed and described herein through the disclosed protein sequence. A wide variety of expression systems may be used to produce UL97 or UL97 homolog peptides as well as fragments, isoforms, and variants.

The nucleic acid sequences provided herein are examples of the genus of nucleic acids and are not intended to be limiting. Also provided are expression vectors comprising these nucleic acids, wherein the nucleic acids are operably linked to an expression control sequence. Further provided are cultured cells comprising the expression vectors. Such expression vectors and cultured cells can be used to make the polypeptides of the invention.

There are a variety of molecules disclosed herein that are nucleic acid based, including for example the nucleic acids that encode UL97, UL97 homologs or fragments or variants thereof. There are a number of compositions and methods which can be used to deliver nucleic acids to cells, either in vitro or in vivo via, for example expression vectors. These methods and compositions can largely be broken down into two classes: viral based delivery systems and non-viral based delivery systems. For example, the nucleic acids can be delivered through a number of direct delivery systems such as, electroporation, lipofection, calcium phosphate precipitation, plasmids, viral vectors, viral nucleic acids, phage nucleic acids, phages, cosmids, or via transfer of genetic material in cells or carriers such as cationic liposomes. Such methods are well known in the art and readily adaptable for use with the compositions and methods described herein. Further, these methods can be used to target certain diseases and cell populations by using the targeting characteristics of the carrier.

As used herein, plasmid or viral vectors are agents that transport the disclosed nucleic acids into the cell without degradation and include a promoter yielding Attorney Docket No. 20674-066WO1

expression of the gene in the cells into which it is delivered. Viral vectors are, for example, Adenovirus, Adeno-associated virus, Herpes virus, Vaccinia virus, Polio virus, AIDS virus, neuronal trophic virus, Sindbis and other RNA viruses, including these viruses with the HIV backbone. Also preferred are any viral families which share the properties of these viruses which make them suitable for use as vectors.

Retroviral vectors, in general, are described by Verma, LM. , Retroviral vectors for gene transfer. In Microbiology-1985, American Society for Microbiology, pp. 229- 232, Washington, (1985), which is incorporated by reference herein. The construction of replication-defective adenoviruses has been described (Berkner et al., J. Virology 61 : 1213-1220 (1987); Massie et al., MoI. Cell. Biol. 6:2872-2883 (1986); Haj-

Ahmad et al., J. Virology 57:267-274 (1986); Davidson et al., J. Virology 61 :1226- 1239 (1987); Zhang BioTechniques 15:868-872 (1993)). The benefit of the use of these viruses as vectors is that they are limited in the extent to which they can spread to other cell types, since they can replicate within an initial infected cell, but are unable to form new infectious viral particles. Recombinant adenoviruses have been shown to achieve high efficiency after direct, in vivo delivery to airway epithelium, hepatocytes, vascular endothelium, CNS parenchyma and a number of other tissue sites. Other useful systems include, for example, replicating and host-restricted non- replicating vaccinia virus vectors. The provided polypeptides or nucleic acids can be delivered via virus like particles. Virus like particles (VLPs) consist of viral protein(s) derived from the structural proteins of a virus. Methods for making and using virus like particles are described in, for example, Garcea and Gissmann, Current Opinion in Biotechnology 15:513-7 (2004). The provided polypeptides can be delivered by subviral dense bodies (DB).

Dense bodies transport proteins into target cells by membrane fusion. Methods for making and using DBs are described in, for example, Pepperl-Klindworth et al., Gene Therapy 10(3):278-84 (2003).

The provided polypeptides can be delivered by tegument aggregates. Methods for making and using tegument aggregates are described in International Publication

NO. WO 2006/110728. Attorney Docket No. 20674-066WO1

II. Compositions

Provided herein are compositions with the provided polypeptides or nucleic acids and a pharmaceutically acceptable carrier. The compositions can also be administered in vivo. The compositions may be administered orally, parenterally (^e-g-_> intravenously), by intramuscular injection, by intraperitoneal injection, transdermally, extracorporeally, topically or the like. The materials may be in solution, suspension (for example, incorporated into microparticles, liposomes, or cells). These may be targeted to a particular cell type via antibodies, receptors, or receptor ligands. By pharmaceutically acceptable is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject, along with the provided polypeptides or nucleic acids, without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. Pharmaceutical carriers are known to those skilled in the art. The carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art. Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.R. Gennaro, Mack Publishing Company, Easton, PA 1995. Typically, an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic. Examples of the pharmaceutically-acceptable carrier include, but are not limited to, saline, Ringer's solution and dextrose solution. Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the polypeptide or nucleic acid, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of agent being administered.

Pharmaceutical compositions may include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice. Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, anti-inflammatory agents, anesthetics, and the like. Attorney Docket No. 20674-066 WOl

Preparations for parenteral administration include sterile aqueous or nonaqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like. A more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. See, e.g., U.S. Patent No. 3,610,795, which is incorporated by reference herein. Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders may be desirable.

Delivery of the pharmaceutical compositions can be carried out via any of several routes of administration, including intramuscular injection, intravenous administration, subcutaneous injection, intrahepatic administration, catheterization (including cardiac catheterization), intracranial injection, nebulization/inhalation or by instillation via bronchoscopy.

The compositions can be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated.

Administration may be topical, oral, by inhalation, or parenterally, for example by intravenous drip, subcutaneous, intraperitoneal or intramuscular injection. The disclosed compositions can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally. Thus, administration of the provided compositions to the brain can be intracranial, subdural, epidural, or intra-cisternal. For example, the provided compositions can be administered by stereotactic delivery. It is also understood that delivery of compositions to the CNS Attorney Docket No. 20674-066 WOl

can be by intravascular delivery if the composition is combined with a moiety that allows for crossing of the blood brain barrier and survival in the blood. Thus, agents can be combined that increase the permeability of the blood brain barrier.

A. Blood-Brain Barrier The UL97, UL97 homolog, fragments or variants of the UL97 or UL97 homolog can be formulated to ensure proper distribution in vivo. To ensure that agents cross the blood brain barrier (BBB), they can be formulated, for example, in liposomes. The liposomes may comprise one or more moieties which are selectively transported into specific cells or organs (targeting moieties), thus providing targeted drug delivery. Exemplary targeting moieties include folate, biotin, mannosides, antibodies, surfactant protein A receptor and gpl20.

To ensure that agents of the invention cross the BBB, they may also be coupled to a BBB transport vector (see Bickel, et al., Adv. Drug Delivery Reviews, vol. 46, pp. 247-279, 2001). Exemplary transport vectors include cationized albumin or the 0X26 monoclonal antibody to the transferrin receptor; these proteins undergo absorptive-mediated and receptor-mediated transcytosis through the BBB, respectively.

Examples of other BBB transport vectors that target receptor-mediated transport systems into the brain include factors such as insulin, insulin-like growth factors (IGF-I, IGF-II), angiotensin II, atrial and brain natriuretic peptide (ANP,

BNP), interleukin I (IL-I) and transferrin. Monoclonal antibodies to the receptors which bind these factors may also be used as BBB transport vectors. BBB transport vectors targeting mechanisms for absorptive-mediated transcytosis include cationic moieties such as cationized LDL, albumin or horseradish peroxidase coupled with polylysine, cationized albumin or cationized immunoglobulins. Small basic oligopeptides such as the dynorphin analogue E-2078 and the ACTH analogue ebiratide can also cross the brain via absorptive-mediated transcytosis and are potential transport vectors.

Other BBB transport vectors target systems for transporting nutrients into the brain. Examples of such BBB transport vectors include hexose moieties such as, for example, glucose; monocarboxylic acids such as, for example, lactic acid; neutral amino acids such as, for example, phenylalanine; amines such as, for example, Attorney Docket No. 20674-066 WOl

choline; basic amino acids such as, for example, arginine; nucleosides such as, for example, adenosine; purine bases such as, for example, adenine, and thyroid hormones such as, for example, triiodothyridine. Antibodies to the extracellular domain of nutrient transporters can also be used as transport vectors. In some cases, the bond linking the agent to the transport vector may be cleaved following transport into the brain in order to liberate the biologically active compound. Exemplary linkers include disulfide bonds, ester-based linkages, thioether linkages, amide bonds, acid-labile linkages, and Schiff base linkages. Avidin/biotin linkers, in which avidin is covalently coupled to the BBB drug transport vector, may also be used. Avidin itself may be a drag transport vector.

B. Dosages

Optimal dosages of compositions depend on a variety of factors. The exact amount required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the disease being treated, the particular virus or vector used and its mode of administration. Thus, it is not possible to specify an exact amount for every composition. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the guidance provided herein.

Effective dosages and schedules for administering the compositions may be determined empirically, and making such determinations is within the skill in the art.

For example, animal models for a variety of protein aggregate disorders can be obtained, for example, from The Jackson Laboratory, 600 Main Street, Bar Harbor, Maine 04609 USA. Both direct (histology) and functional measurements (learning ability, memory skills, neurologic scores and the like) can be used to monitor response to therapy. These methods involve the sacrifice of representative animals to evaluate the population, increasing the animal numbers necessary for the experiments.

The dosage ranges for the administration of the compositions are those large enough to produce the desired effect in which the symptoms of the disease are affected. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions and anaphylactic reactions. The dosage can be adjusted by the individual physician in the event of any counterindications. Dosage can vary, and Attorney Docket No. 20674-066 WOl

can be administered in one or more dose administrations daily, for one or several days.

C. Combination Therapy

The provided compositions may be used alone or in combination with one or more additive compounds or therapeutic agent. The compound or therapeutic agent may be any compound or substance known in the art which may be beneficial to the subject. The second compound may be any compound which is known in the art to treat, prevent, or reduce the symptoms of a protein aggregation disorder. Furthermore, the second compound may be any compound of benefit to the subject when administered in combination with the administration of a compound of the invention, e.g. a neuroprotective compound. The language in combination with a second compound or therapeutic agent includes co-administration of the compositions, as well as sequential administration. Thus, the second composition or therapeutic agent can be administered prior to, along with or after, the first compositions. Therapeutic agents that may be administered in combination with the provided compositions may be effective in controlling detrimental protein aggregate deposition either following their entry into the brain (following penetration of the blood brain barrier) or from the periphery. When acting from the periphery, a therapeutic agent may alter the equilibrium of a protein between the brain and the plasma so as to favor the exit of the protein from the brain. An increase in the exit of the protein from the brain would result in a decrease in the protein brain concentration and therefore favor a decrease in protein deposition in aggregates. Alternatively, therapeutic agents that penetrate the blood brain barrier could control deposition by acting directly on brain proteins, for example, by maintaining it in a non-fibrillar form or favoring its clearance from the brain.

Therapeutic agents for use in the provided methods include, but are not limited to, chemotherapeutic agents, anti-inflammatory agents, anti-viral agents, anti- retroviral agents, anti-opportunistic agents, antibiotics, anticonvulsants, immunosuppressive agents, apoptosis-inducing agents, lazaroids, bioenergetics, antipsychotics, N-methyl D-aspartate (NMDA) antagonists, dopamine antagonists, antidepressants, acetylcholinesterase inhibitors, cholinesterase inhibitors, antiglutamatergic agents, dopamine receptors, dopamine agonists, immunoglobulins Attorney Docket No. 20674-066 WOl

and pain medications. Thus, the therapeutic agent can be levodopa, carbidopa, benserazide, gingko biloba, qigong tolcapone, entacapone, bromocriptine, pergolide, pramipexole, ropinirole , cabergoline, apomorphine, lisuride, selegiline, rasafϊline, quetiapine, rivastagime, tramiprosate, xaliproden, R-flurbiprofen or leuprolide. III. Protein Aggregate Diseases

As used herein the terms treatment, treat or treating refer to a method of reducing the effects of a disease or condition or symptom of the disease or condition. Thus in the disclosed method treatment can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% reduction in the severity of an established disease or condition or symptom of the disease or condition. For example, the method for treating a protein aggregate disorder is considered to be a treatment if there is at least a 10% reduction in one or more symptoms of the disease in a subject as compared to control. Thus the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100% or any percent reduction in between 10 and 100 as compared to native or control levels. It is understood that treatment does not necessarily refer to a cure or complete ablation of the disease, condition or symptoms of the disease or condition.

As used herein, the terms prevent, preventing and prevention of a disease or disorder refers to an action, for example, of administration of a therapeutic agent, that occurs before a subject begins to suffer from one or more symptoms of the disease or disorder, which inhibits or delays onset of the severity of one or more symptoms of the disease or disorder.

As used herein, subject can be a vertebrate, more specifically a mammal (e.g., a human, horse, pig, rabbit, dog, sheep, goat, non-human primate, cow, cat, guinea pig or rodent), a fish, a bird or a reptile or an amphibian. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered. As used herein, patient or subject may be used interchangeably and can refer to a subject afflicted with a disease or disorder. The term patient or subject includes human and veterinary subjects.

Protein aggregate diseases or disorders can be treated or prevented using the methods and compositions provided herein. As used herein, a protein aggregate disease or disorder includes a disease, disorder or condition that is associated with detrimental protein aggregation in a subject. Detrimental protein aggregation is the Attorney Docket No. 20674-066WO1

undesirable and harmful accumulation, oligomerization, fibrillization or aggregation, of two or more, hetero- or homomeric, proteins or peptides. A detrimental protein aggregate may be deposited in bodies, inclusions or plaques, the characteristics of which are often indicative of disease and contain disease-specific proteins. A detrimental protein aggregate is a three dimensional structure that may contain, for example, misfolded protein composed of β-sheets, fibril-like structures and/or highly hydrophobic domains that tend to aggregate and are toxic to cells. Furthermore, a detrimental protein aggregate may be described as amyloid-like, although it does not contain amyloid deposits and is not considered to be associated with an Amyloidosis as it does not adhere to the strict definition of amyloid, i.e., it does not display red- green or apple-green birefringence under polarized light following staining with Congo red.

Protein aggregation diseases include, but are not limited to a disease characterized by amyloidosis, sickle cell disease, a prion disease, a polyglutamine repeat disease, a disease characterized by α-synuclein aggregation, a disease characterized by SODl aggregation and a protein aggregated myopathy.

Protein aggregate myopathies include, but are not limited to, a desmin-related myopathy, an inclusion body myopathy, an actinopathy or a myosinopathy. Protein aggregate diseases include neurodegenerative diseases. Neurodegenerative diseases include, but are not limited to, a prion disease,

Alzheimer's disease, Pick's disease, progressive supranuclear palsy, frontotemporal dementia, corticobasal degeneration, postencephalitic parkinsonism, Parkinson's disease, multiple system atrophy, Huntington's disease, Batten disease, dementia with Lewy bodies, Hallervorden-Spatz syndrome and amyotrophic lateral sclerosis disease. Prion diseases include, but are not limited to, Creutzfeldt- Jakob disease, bovine spongiform encephalopathy, a spongiform encephalopathy, kuru, scrapie, chronic wasting disease, fatal familial insomnia, Alper's syndrome, Grestmann- Straussler-Scheinker syndrome, transmissible mink encephalopathy, feline spongiform encephalopathy and exotic ungulate encephalopathy. Diseases characterized by amyloidosis include, but are not limited to, systemic amyloidosis, familial or hereditary amyloidosis, organ-specific amyloidosis, AL amyloidosis, AA amyloidosis, gelsolin amyloidosis, Appalachian type amyloidosis, Attorney Docket No. 20674-066 WOl

Shar Pei fever, diabetes mellitus type 2, cardiac amyloidosis, inclusion body myositis, congophilic angiopathy and Down's syndrome. The disease characterized by amyloidosis also includes diseases associated with a mutation in a gene such as, for example, apolipoprotein Al, lysozyme, transthyretin, apolipoprotein B and fibrinogen.

Protein aggregation disorders or proteopathies also include, but are not limited to protein conformational disorders, polyglutamine diseases, serpinopathies, tauopathies, dystrophia myotonica, dentatorubro-pallidoluysian atrophy (DRPLA), Friedreich's ataxia, fragile X syndrome, fragile XE mental retardation, Machado- Joseph Disease (MJD or SCA3), spinobulbar muscular atrophy (also known as

Kennedy's Disease), spinocerebellar ataxia type 1 (SCAl) gene, spinocerebellar ataxia type 2 (SCA2), spinocerebellar ataxia type 6 (SCA6), spinocerebellar ataxia type 7 (SCA7), spinocerebellar ataxia type 17 (SCAl 7), chronic liver diseases, Amyotrophic Lateral Sclerosis (ALS), haemolytic anemia, cystic fibrosis, Wilson's Disease, neurofibromatosis type 2, demyelinating peripheral neuropathies, retinitis pigmentosa,

Marfan syndrome, emphysema, idiopathic pulmonary fibrosis, Argyophilic grain dementia, diffuse neurofibrillary tangles with calcification, or subacute sclerosing panencephalitis.

Polyglutamine diseases include, but are not limited to, dystrophia myotonica, dentatorubro-pallidoluysian atrophy (DRPLA), Friedreich's ataxia, fragile X syndrome, fragile XE mental retardation, Machado-Joseph disease, spinobulbar muscular atrophy (also known as Kennedy's Disease), spinocerebellar ataxia and Huntington's disease (HD).

Protein aggregate diseases also include lysosomal storage diseases. Lysosomal storage diseases include lipid storage diseases, leukodystrophies, mucopolysaccharidoses, glycoprotein storage diseases and mucolipidoses. Lysosomal storage diseases are characterized by the accumulation of, for example, lipids, carbohydrates, glycoproteins, and glycosaminoglcans in some of the body's cells and tissues. Lipid storage diseases include, Sandhoff disease, Tay-Sachs disease, mucolipidosis type IV, Gaucher disease, Niemann-Pick disease, Fabry diseases,

Krabbe disease, gangliodisoses, metachromatic leukodystrophy and Wolman's disease. Attorney Docket No. 20674-066WO1

Disclosed are materials, compositions, and components that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed methods and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a biomarker is disclosed and discussed and a number of modifications that can be made to a number of molecules including the biomarker are discussed, each and every combination and permutation of the biomarker and the modifications that are possible are specifically contemplated unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D, is disclosed, then even if each is not individually recited, each is individually and collectively contemplated. Thus, is this example, each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. Likewise, any subset or combination of these is also specifically contemplated and disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods, and that each such combination is specifically contemplated and should be considered disclosed.

Ranges may be expressed herein as from about one particular value, and/or to about another particular value. Similarly, when values are expressed as approximations, by use of the term about, it will be understood that the particular value is included. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. Attorney Docket No. 20674-066WO1

Optional or optionally means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. For example, the phrase optionally the composition can comprise a combination means that the composition may comprise a combination of different molecules or may not include a combination such that the description includes both the combination and the absence of the combination (i.e., individual members of the combination).

Publications cited herein and the material for which they are cited are hereby specifically incorporated by reference in their entireties. It is to be understood that the disclosed method and compositions are not limited to specific synthetic methods, specific analytical techniques, or to particular reagents unless otherwise specified, and, as such, may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. Note the headings used herein are for organizational purposes only and are not meant to limit the description provided herein or the claims attached hereto.

EXAMPLES

Example 1. UL97 Kinase Activity Required for Hyperphosphorylation of Rb and Inhibition of Nuclear Aggresomes MATERIALS AND METHODS

Cells and Viruses. Human foreskin fibroblast (HFF) cells were established and routinely propagated in monolayer cultures in minimum Eagle's medium with Earle's salts supplemented with 10% fetal bovine serum (FBS), 2mM L-glutamine, 100 μg/ml penicillin G and 25 μg/ml gentamicin. HCMV strain AD 169 was obtained from the American Type Culture Collection (Manassas, VA) and virus stocks were prepared and titered as described previously (Prichard et al., Antimicrob. Agents Chemother. 50:1336-41 (2006)). The construction and characterization of a UL97 null mutant (RCΔ97.08) was described previously (Prichard et al., J. Virol. 73:5663-70 (1999). Maribavir was obtained through the Antiviral Substances Program of the National Institute for Allergy and Infectious Diseases.

Plasmids. Construction of the plasmids expressing the pp65-GFP fusion protein as well as those for the epitope tagged versions of the wt UL97 ORF and the K355M Attorney Docket No. 20674-066 WOl

mutation were described previously (Prichard et al., J. Virol. 79:15494-502 (2005)). Mutation of the LxCxD (SEQ ID NO:2) motif was performed by amplifying the UL97 ORF of AD169 using UL97 forward primer 5'-CAC CAT GTC CTC CGC ACT TCG GTC T-3' (SEQ ID NO: 14) and LxCxD (SEQ ID NO:2) reverse primer 5'-TCA CCT TCG ACC GCC CGT AGC TGT CGA TG-3 ' (SEQ ID NO: 15), and UL97 reverse primer 5'-TTA CTC GGG GAA CAG TTG G-3' (SEQ ID NO: 16) with LxCxD (SEQ ID NO:2) forward primer 5'-AGT GGAAGC TGG CGG GCATCG ACA GCT AC-3 ' (SEQ ID NO: 17). The two PCR products were gel purified, mixed in an equimolar ratio and used as a template for PCR using the UL97 forward primer and UL97 reverse primer. The PCR product was cloned into the pENTR/D/TOPO vector (Invitrogen, Carlsbad, CA) to yield pMP263. The C428G mutation was confirmed by sequencing and recombined into pcDNA3 V5-DEST (Invitrogen, Carlsbad, CA) to yield pMP 264. Mutation of the LxCxE (SEQ ID NO:1) motif was performed in a similar manner using the UL97 forward primer with LxCxE reverse primer 5 '-GGT GCC GAA CGC GCC GGC GCT TTG AAG-3 ' (SEQ ID NO: 18), and UL97 reverse primer with LxCxE forward primer 5'-CCA CGG CTT GCG CGG CCG CGAAAC TTC-3' (SEQ ID NO: 19). The two PCR products were combined and used as a template for a second PCR using the UL97 forward primer and UL97 reverse primer. The product was cloned into pENTR/D/TOPO (pMP 265) and the C 151 G mutation was confirmed by sequence analysis. The open reading frame containing the mutation was them recombined into the pcDNA3 V5-DEST vector to yield pMP 269.

Polyacrylamide gels and western blotting. HFF cells were infected at a multiplicity of infection of 2 PFU/cell and at 24 and 72 hours following infection they were disrupted in 2X Lamelli buffer (Sigma- Aldrich, St. Louis, MO) and separated on

10%, 7.5%, or 5% polyacrylamide gels depending on the size of the protein to be resolved (BioRad, Hercules, CA). Separated proteins were transferred to PVDF membranes (Roche Applied Science, Indianapolis, IN) in a buffer containing 28 mM Tris, 39 mM glycine, 0.0375 % SDS and 20% methanol in a semi dry transfer cell (BioRad, Hercules, CA). Membranes were blocked in 1% blocking buffer (Roche

Applied Science, Indianapolis, IN), incubated with primary antibodies overnight at 4°C and washed extensively with PBS supplemented with 0.02% tween 20. A Attorney Docket No. 20674-066 WOl

secondary antibody conjugated to alkaline phosphatase (Southern Biotechnology Associates, Birmingham, AL) and CDP* (Roche Applied Science, Indianapolis, ESf) were used to detect the bound primary antibody. Monoclonal antibodies used in these studies were directed against PML (H-238) (Santa Cruz Biotechnology, Santa Cruz, CA), DAXX (Upstate Cell Signaling Solutions, Tamecula, CA), p53, retinoblastoma, β-tubulin, CREB binding protein (CBP) (Chemicon International, Tamecula, CA), and both V5 and Xpress (Invitrogen, Carlsbad, CA).

Indirect immunofluorescence microscopy. Cells expressing proteins from transfected plasmids were visualized by methods published previously (Prichard et al., J. Virol. 79: 15494-502 (2005)). Briefly, monolayers of HFF cells were grown on 12 mm diameter coverslips in 24-well plates. Transfected cells were fixed for 15 minutes with freshly prepared 2% formaldehyde in phosphate buffered saline (PBS), washed two times with PBS, and membranes were permeabilized with 0.2 % Triton X-100 in PBS for 15 minutes. Monoclonal antibody to the V5 epitope tag was purchased from Invitrogen (Carlsbad, CA). Monoclonal antibodies to IEl (63-27) and ppUL44 (28-

21) were generously provided by Bill Britt and were used as culture supernatants with goat anti-mouse secondary antibodies conjugated to FITC or Texas Red (Southern Biotechnology, Birmingham, AL). Isolation of nuclear and cytoplasmic aggresomes. Low passage HFF cells were infected with RCΔ97.08 at a low MOI in 175 cm² flasks, and infected cells were passaged at 7 days post infection as plaques started to form as well as 12 and 16 dpi until 100% CPE was observed. Infected cells were dislodged with 0.25% trypisn- EDTA (Gibco, Grand Island, NY) and resuspended in a volume of 10 ml growth medium. Cells were collected by centrifugation and resuspended in PBS with the addition of 0.6 % NP-40 and nuclei were centrifuged through a cushion of histopaque

1077 (Sigma Chemical Company, St. Louis, MO) at 1000 x g for 5 minutes. Nuclei were lysed in PBS supplemented with 2.5 M NaCl and cellular DNA was degraded with 10,000 units of deoxyribonuclease I. An equal volume of 5 M urea was added to the nuclear lysate and the nuclear aggresomes were collected by centrifugation at 3500 x g for 10 minutes through a histopaque cushion. Nuclear aggresomes were resuspended in PBS supplemented with 0.5% NP-40 and frozen at -80 ⁰C. Cytoplasmic aggresomes were isolated from the cytoplasmic fraction by Attorney Docket No. 20674-066WO1

sedimentation at 3500 x g. The sedimented material was resuspended in a PBS buffer containing 5M urea and aggresomes were collected by sedimentation through a histopaque cushion and resuspended in PBS with 0.5% NP-40 and frozen at -80 ⁰C. Immunoprecipitation. COS7 cells in 75cm² flasks were transfected with plasmids expressing epitope tagged versions of pUL97 and two negative controls, UL89 and

UL104. Two days post transfection cells were collected by centrifugation and lysed in 1 ml RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% deoxycholate, 0.1% SDS, 50 niM Tris pH 7.5). Cell lysates were clarified at 10,000 x g and 50 μL of Protein A sepharose bound to the V5 monoclonal antibody was added to each supernatant. The suspension was incubated for 12 h at 4°C, the beads were washed twice with RIPA buffer and frozen until further use. Bound proteins on 10 μl of beads were added to a tube containing 100 μl of PBS and 25 μg of a recombinant protein consisting of the carboxyl terminus of Rb fused to the maltose binding protein was added to each tube (Cell Signaling Technology, Beverly MA). The suspension was incubated for 1 h at 37°C and the beads were washed three times in PBS. Proteins bound to the beads were eluted with Lamelli buffer, separated on 7.5 % SDS PAGE gels and transferred to a PVDF membrane. Proteins were detected with the monoclonal antibody to the V5 epitope and also with a rabbit polyclonal antisera directed against Rb (Neomarkers, Freemont, CA). Tryptic Digestion of Viral Inclusions. HCMV inclusion bodies were denatured by the addition of urea to 8M and heating to 37°C for 30 min. The sample was then diluted 4-fold with 100 mM ammonium bicarbonate (AB) and CaC12 was added to ImM. Methylated, sequencing-grade porcine trypsin (Promega, Madison, WI) was added at a substrate-to-enzyme ratio of 50:1 (mass:mass) and incubated at 37°C for 15 hours. Sample cleanup was achieved using a 1-mL SPE Cl 8 column (Supelco,

Bellefonte, PA). The peptides were eluted from each column with 1 mL of methanol and concentrated via SpeedVac. The samples were reconstituted to 10 μg/μL with 25 mM ammonium bicarbonate and frozen at -20⁰C until analyzed. Tandem Mass Spectrometric Analysis of Peptides. Peptide samples were analyzed by reversed phase cLC coupled directly with electrospray tandem mass spectrometers

(Thermo Finnigan, models LCQ™ Duo and LCQ™ DecaXP, San Jose, CA). Chromatography was performed on a 60-cm, 150 μm i.d. X 360 μm o.d capillary Attorney Docket No. 20674-066WO1

column (Polymicro Technologies, Phoenix, AZ) packed with Jupiter C18 5-μm- diameter particles (Phenomenex, Torrence, CA). A solvent gradient was used to elute the peptides using 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). The gradient was linear from 0 to 5% solvent B in 20 minutes, followed by 5 to 70% solvent B in 80 minutes, and then 70-85% solvent B in 45 minutes. Solvent flow rate was 1.8 μl/min. The capillary LC system was coupled to a LCQ ion trap mass spectrometer (Thermo Finnigan, San Jose, CA) using an in-house manufactured ESI interface in which no sheath gas or makeup liquid was used. The temperature of heated capillary and electrospray voltage was 200oC and 3.0 kV, respectively. Samples were analyzed using the data-dependent MS/MS mode over the m/z range of

300-2000. The three most abundant ions detected in each MS scan were selected for collision-induced dissociation.

SEQUEST Analysis. The SEQUEST algorithm was run on each of the datasets against a combined database comprised of the HCMV.fasta and the human, fasta from the National Center for Biotechnology Information. Tandem MS peaks were generated by extract_msn.exe, part of the SEQUEST software package. A peptide was considered to be a match by using a conservative criteria set developed by Yates and coworkers (Link et al, 1999; Washburn et al., 2001). Briefly, all accepted SEQUEST results had a delta Cn of 0.1 or greater. Peptides with a +1 charge state were accepted if they were fully tryptic and had a cross correlation (XCorr) of at least

1.9. Peptides with a +2 charge state were accepted if they were fully tryptic or partially tryptic and had an XCorr of at least 2.2. Peptides with +2 or +3 charge states with an XCorr of at least 3.0 or 3.75, respectively, were accepted regardless of their tryptic state. RESULTS

Aggregation of pp65 is affected by pp71 and IEl. Inappropriate aggregation of viral proteins occurs in the absence of the UL97 kinase. It was reported previously, that expression of a pp65-GFP fusion protein in COS7 cells was sufficient to induce formation of large nuclear aggregates, which were inhibited by the coexpression of pUL97 (Prichard et al., J. Virol. 79: 15494-502 (2005)). Expression plasmids for 128 viral open reading frames were individually cotransfected with a plasmid expressing GFP-tagged pp65 and the effect on aggregation was assessed by immunofluoresence Attorney Docket No. 20674-066 WOl

microscopy. Plasmids expressing either pp71 or IEl significantly increased the formation of pp65 aggregates and did not appear to be related to expression levels since cotransfection with a plasmid expressing both IEl and IE2 had no effect. Confirmatory transfections demonstrated that expression of both IEl and pp71 significantly increased the proportion of pp65 expressing cells that exhibited more than two large nuclear aggregates (Fig. 1). Both proteins have been reported to interact with components of PML domains and also interact functionally with Rb. PML bodies have also been reported to be involved in protein aggregation through the formation of nuclear aggresomes, which are cellular structures thought to be involved in the sequestration of misfolded proteins. These data taken together indicate that

UL97 affects aggregate formation by altering cellular functions associated with PML bodies.

Sequestration of proteins in nuclear aggresomes is inhibited by the kinase.

Reports linking PML bodies and the nuclear aggregation prompted an examination of the effect of the UL97 kinase on the aggregation of two other proteins in the nucleus.

The pp71 tegument protein was reported to form large nuclear aggregates similar to those formed by pp65 and was affected by the presence of PML. A cellular protein, GFP-GCP 170*, also induces large aggregates called nuclear aggresomes and been used as a marker for these structures. Transfection of COS7 cells with epitope tagged versions of pp71 confirmed that it formed large nuclear aggregates (Fig. 2B) that resembled those formed by pp65 (Fig. 2A), and cytoplasmic aggregations were also observed that contained pp71. The expression of GFP-GCP 170* was also sufficient to induce cytoplasmic and nuclear aggresomes in COS7 (Fig. 2C). The nuclear aggregates with the viral proteins and pp65 were morphologically similar to the nuclear aggresomes induced by GFP-GCP 170* and the cytoplasmic aggregates induced with pp71 also resembled the cytoplasmic aggresomes. To characterize the effect of the kinase on the aggregation of these proteins, the kinase and a K355M mutant without enzymatic activity were coexpressed with ρp65, pρ71 and GFP- GCP170*. The kinase inhibited the formation of pp65 aggregates (Fig. 2 D-F), whereas the kinase negative form of UL97 (K355M) was unable to inhibit their formation and was recruited to the large nuclear aggregates (Fig. 2 G-I). The inhibition of aggregation formation by the kinase was also antagonized by Maribavir Attorney Docket No. 20674-066 WOl

(MBV), a specific inhibitor of its kinase activity. The kinase also inhibited the aggregation of pp71 in the nucleus of cotransfected cells and both proteins remained in the cytoplasm (Fig. 2 J-L). The K355M mutant was unable to inhibit the aggregation of pp71 and was recruited to both nuclear and cytoplasmic aggregates (Fig. 2 M-O). The kinase was also unable to inhibit the aggregation of pp71 in the presence of MBV, confirming that the inhibition was dependent on its enzymatic activity.

These results indicate that the ability of the kinase to inhibit nuclear aggregates was not limited to pp65 but also inhibited the aggregation of another viral phosphoproteins. The kinase effect on general cellular function and on nuclear aggresomes induced by GFP-GCP 170* was examined. Cotransfection with plasmids expressing the kinase reduced the size and number of both nuclear and cytoplasmic aggresomes and also affected their distribution in the cell (Fig. 2 P-R). This activity appeared to be dependent on its kinase activity since the K355M mutant failed to inhibit their formation (Fig. 2 S-T) and rather, was recruited to cytoplasmic aggresomes. Similar results were also observed when the active kinase was inhibited with MBV. These data indicate that the kinase inhibits nuclear aggregation and the formation of both cytoplasmic and nuclear aggresomes. They indicate also, that the aggregation of the virion tegument proteins in the nucleus relates to the formation of nuclear aggresomes.

Tegument aggregates are nuclear aggresomes containing virion proteins. One interpretation of the data was that virion tegument proteins expressed in uninfected cells were inside nuclear aggresomes and that their disruption by the kinase prevented the cell from sequestering these proteins. To test this hypothesis, GFP-GCP 170* was co-expressed with virion tegument proteins to determine if they colocalized. Each of the tegument proteins, pp65, pp71, and pUL69 localized with GFP-GCP 170*, indicating that the aggregation observed with these proteins was mediated by the aggresomes. Therefore, the structures referred to as tegument aggregates are nuclear aggresomes that contain proteins. The data also indicate that inhibition of tegument protein aggregation is related to the ability of the kinase to prevent the formation of these cellular structures. Attorney Docket No. 20674-066 WOl

Aggresomes contain large quantities of virion proteins. To determine the protein content of the HCMV nuclear and cytoplasmic aggresomes, they were purified from infected cells by methods described previously and subjected to an analysis by mass spectrometry (Prichard et al., J. Virol. 79:15494-502 (2005); Varnum et al, J. Virol. 78: 10960-6 (2004)). The aggresomes were denatured and digested with trypsin. To identify the viral proteins found in the aggresomes, the complex mixture of peptides was analyzed by two-dimensional liquid chromatography coupled to MS/MS and the results were compared to a HCMV-FASTA database. This analysis revealed that the aggresomes contained large quantities of viral structural proteins (Table 2).

Table 2. Viral Proteins Identified by LC/MS/MS in Cytoplasmic and Nuclear

Aggresomes.

Cytoplasmic Nuclear Aggresomes Aggresomes

HCM Max No. of Max No. of Description

V -^corr unique Xcorr unique

ORF Peptides Peptides

IRSl 5.12 4.73 Transcriptional transactivator;

US22 family member

UL25 4.97 15 5.54 25 Tegument protein; UL25 family member

UL26 3.46 2 4.91 4 Tegument protein; US22 family member

UL31 N.D. N>D. 3.97 3 Hypothetical protein

UL32 4.71 4 4.38 4 Pp 150 tegument protein

UL34 N.D. N.D. 4.34 2 Transcriptional repressor

UL35 N.D. N.D. 6.58 3 UL25 family member

UL44 5.57 11 5.16 7 Processivity subunit of DNA polymerase; HSV-I UL42 counterpart

UL46 N.D. N.D. 4.18 3 Intercapsomeric triplex capsid protein; HSV-I UL38 counterpart

UL47 N.D. N.D. 3.50 1 Tegument protein; HSV-1UL37 counterpart

UL48 4.30 1 5.68 5 Tegument protein; HSV-I UL36 counterpart

UL48A 7.17 5 7.03 2 Capsid protein located at tips of hexons; HSV-I UL35 counterpart

UL50 6.23 1 N.D. N.D Membrane-associated protein involved in nuclear capsid egress;

HSV-I UL34 counterpart Attorney Docket No. 20674-066 WOl

UL57 1.91 1 N.D. N.D. Single-stranded DNA-bmdnig protein; HSV-I UL29 counterpart

UL69 N.D. N.D. 3.28 2 Post-transcriptional regulator of gene expression; HSV-I UL54 counterpart

UL71 5.09 2 N.D. N.D. Tegument protein; HSV-I U151 counterpart

UL77 N.D. N.D. 4.59 1 DNA packaging protein; HSV-I

UL25 counterpart

UL80 5.60 9 4.10 2 Protease (N-terminus) and minor scaffold protein (C -terminus);

HSVO-I UL26 counterpart

UL82 N.D. N.D. 4.82 5 Pp71 upper matrix phosphosprotein; tegument protein; transactivator of MIEP

UL83 6.00 60 6.56 82 Pp65 lower matrix phosphosprotein; tegument protein

UL84 5.05 4 5.65 3 Transdominant inhibitor of IE2- mediated transactivation

UL85 3.00 2 2.99 3 Intercapsomeric triplex capsid protein; HSV-I UL 18 coutnerpart

UL94 N.D. N.D. 3.83 1 Tegument protein; HSV-I ULl 6 counterpart

UL98 N.D. N.D. 3.32 1 DNase; HSV-I UL 12 coutnerpart

ULl 04 N.D. N.D. 3.84 2 DNA packaging protein; capsid portal protein; HSV-I UL16 counterpart

UL112 1.97 1 N.D. N.D. Hypothetical protein

ULl 15 4.16 1 N.D. N.D. Envelope glycoprotein; associated with gH and gθ; HSV-I ULl counterpart; gL

ULl 22 4.24 3 3.23 1 Immediate-early transcriptional regulator; IE2

US22 2.48 1 3.53 1 Tegument protein; US22 family member

N.D., not detected

There were 25 HCMV proteins in the nuclear aggresomes and 19 in the cytoplasmic aggresomes. These include the capsid proteins UL46 (minor capsid binding protein), UL48A (smallest capsid protein), UL80 (assembly protein), UL85 (minor capsid protein), and UL86 (major capsid protein), as well as a number of tegument proteins including UL25, UL26, UL32, UL35, UL47, UL48, UL82, UL83, UL94, and US22. In addition, a number of proteins involved in transcription and DNA replication were also present including IRSl, UL31, UL34, UL44, UL57, UL69, Attorney Docket No. 20674-066 WOl

UL84, UL98, UL104, and UL122. Overall the ratios of viral proteins present in the aggresomes resemble dense bodies, rather than virions, showing that they are incorporated prior to genome packaging.

To identify the cellular proteins present in the aggresomes, the result from the mass spectroscopy analysis were compared to the predicted peptides of a human- FASTA database. The nuclear and cytoplasmic aggresomes contained a number of cellular heat shock proteins including HSP70, HSP71c, HSP70-2, and HSP-60 (Table 3).

Table 3. Notable Cellular Proteins Identified by LC/MS/MS in Cytoplasmic and Nuclear Aggresomes.

Reference Description Max

Λcorr gil5729877 Heat shock 7OkD protein 10 (HSC71) 4.74 gil4758570 Heat shock 7OkD protein 9B 5.11 gill 708307 Heat shock-related 70 kD protein 2 3.94 gill29379 Mitochondrial matrix protein pi precursor 3.93

Cytoplasmic (HSP-60) Aggresomes gill 25731 ATP-dependent DNA Helicase II, 80 kDa 7.14 subunit gill 14762 Nucleophosmin (nucleolar phosphoprotein 6.12

B23) gil7446411 Aurora-related kinase 1 2.29 gil5729877 Heat shock 7OkD protein 10 (HSV71) 6.53 gil4502549 Calmodulin 2 (phosphorylase kinase, 4.84

Aggresomes delta) gil2119712 dnaK-type molecular chaperone HSPAlL 3.21

The heat-shock proteins are known to be associated with aggresomes, however, their exact role in their formation is still unclear. The cellular protein aurora-related kinase 1 was present in the nuclear aggresomes, which is involved in chromosome segregation and may act during viral DNA replication. Nucleophosmin was detected in the cytoplasmic aggresomes and not in the nuclear-derived aggresomes. Nucleophosmin is a nucleolar protein that is critical for centresome duplication and genomic stability. Overexpression of nucleophosmin has been noted in a number of malignancies, which may be attributed to its ability to inactivate p53; thus suppressing apoptosis. The inactivation of p53 has been noted in HCMV infected Attorney Docket No. 20674-066WO1

cells although it was thought that this might only be due to the viral immediate early proteins.

PML bodies are affected by the kinase. Nuclear aggresomes form in a dynamic microtubule dependent process that initiates with fusion of small aggregations located at or near PML bodies. To determine the effect of pUL97 on these structures, this and other proteins were expressed in COS7 cells and visualized PML bodies with an antibody to SPlOO, which is a marker of these domains. IEl has been reported to disperse nuclear structures and reduced their number significantly (Fig. 3B), while the over expression of another viral nuclear protein (ppUL44, ICP36) can not (Fig. 3A). Their number was also unaffected by the expression of pp71 , however it colocalized with these structures and is consistent with previous reports. The localization of pp71 near these structures is also consistent with early processes in the formation of nuclear aggresomes and large pp71 aggresomes are frequently observed adjacent to them. Expression of pUL97 also significantly reduced the number of PML bodies (Fig. 3D) in a manner that was kinase dependent since neither the K355M mutant, or pUL97 in the presence of maribavir could affect their numbers (Fig. 3E,F). While this effect was not as robust as the dispersion by IEl, it was both significant and repeatable. These results indicated that the effect of the kinase on PML bodies might be related to its ability to inhibit nuclear aggresome formation. UL97 kinase activity is required for the hyperphosphorylation of Rb. The ability of the kinase to disperse PML bodies suggested that it might be affecting one of the proteins associated with this complex. Proteins in this complex were examined by Western blots using samples derived from HFF cells that were uninfected, infected with AD 169 a recombinant virus (RCΔ97) containing a large deletion in UL97 at a multiplicity of infection of 3 PFU/cell. Some of the infections were conducted in the presence of MBV to confirm that the observed changes were due to a deficiency of its kinase activity. No changes were observed in the quantity or sumoylation of PML, CPB or DAXX (Fig. 4). In cells infected with the wt virus, increases in levels of p53 were observed (Fig. 4 lanes 9-12), but this increase was unaffected by the deletion of the UL97 open reading frame or treatment with maribavir. Also consistent with this previous report was an increase of hyperphosphorylated Rb at both 24 and 72 hours following infection (Fig. 4, lanes 3,9). This did not occur at 24 hours post infection in Attorney Docket No. 20674-066 WOl

cells infected with the wt virus that were treated with maribavir (MBV) (Fig. 4 lane 4), or in cells infected with RCΔ97 with or without MBV (Fig. 4 lanes 5,6). Similarly, at 72 hours after infection, cells infected with the wt virus had high levels of hyperphsophorylated Rb that was significantly reduced with the addition of MBV (Fig. 4 lanes 9,10) and levels remained low in cells infected with RCΔ97 (Fig. 4 lane

11). Cells infected with the UL97 deletion virus had reduced levels of hyperphosphorylated Rb that was relatively unaffected by the addition of MBV (Fig. 4, compare lane 9 with 11,12). Taken together, these data confirm that HCMV infection increases levels of hyperphosphorylated Rb and indicate that UL97 kinase activity is required for this effect. Rb interacts directly with PML and has been shown to increase the number of PML bodies. A previous report has shown that pp71 directs the proteasome dependent degradation of hypophosphorylated Rb, however observations presented here are distinct in that the hyperphosphorylation and stabilization of this form of Rb is inefficient in the absence UL97 kinase activity. UL97 kinase contains two Rb binding motifs. The amino acid sequence of UL97 kinase was examined for Rb binding motifs. Two binding domains were identified (Fig. 5A). The amino terminus contains a consensus LxCxE (SEQ ID NO:1) motif and an adjacent DSSE (SEQ ID NO: 13) motif conserved among proteins that bind Rb including SV40 large T antigen, adenovirus ElA, and HPV E7. The second motif, closer to the carboxyl terminus contains an LxCxD (SEQ ID NO:2) sequence, and is similar to the amino acid sequence required for Rb binding in pp71. The amino terminus of the chimpanzee cytomegalovirus UL97 homolog does not share significant identity with UL97 until the LxCxE (SEQ ID NO: 1) motif and DSSE (SEQ ID NO: 13) motifs, which are well conserved, as is the LxCxD (SEQ ID NO:2) motif. The UL97 homologs in HHV6-A, HHV6-B, and HHV7 U69, do not share significant amino acid identity with the amino terminus of UL97, yet all retain the LxCxE (SEQ ID NO:1) motif and the DSSE (SEQ ID NO: 13) motif is conserved in all the betaherpesviruses except HHV7. The kinase domains of U69 and UL97 are more highly conserved; however the other betaherpesviruses do not retain a conserved the LxCxD (SEQ ID NO:2) motif. The conserved Rb binding motifs in the kinase homologs of the betaherpesviruses are consistent with its affects on Rb and indicate that this function is important in the replication of these viruses. Attorney Docket No. 20674-066 WOl

To explore Rb binding activity further, epitope tagged versions of the kinase were expressed in COS7 cells and immunopreciptiated with a monoclonal antibody to the V5 epitope. Proteins bound to protein A sepharose beads were incubated with recombinant Rb protein to investigate a potential interaction. Expression of the kinase, as well as the K355M, C151G, C428G mutants were easily detectable using a monoclonal antibody to the epitope tags, as was the expression of two control proteins pUL89 and pUL104 (Fig. 5B). The K355M mutation eliminates kinase activity, while the C151G and C428G mutations disrupt the LxCxE (SEQ ID NO:1) and LxCxD (SEQ ID NO: 2) motifs, respectively. Recombinant Rb was specifically precipitated by beads bound to each of the pUL97 proteins, but not to beads bound to either of the negative control proteins. Neither C151G nor C428G appeared to be sufficient to eliminate Rb binding. A similar result was described for BRCAl where disruption of the LxCxE (SEQ ID NO:1) motif did not disrupt Rb binding, but impaired the inactivation of Rb. Nevertheless, these results indicate that Rb can be co-precipitated by the kinase and is consistent with a direct interaction between the kinase and Rb.

Conserved LxCxE (SEQ BO NO:1) Rb binding motif is required for the inhibition of aggresome formation. Initial studied studies suggested the kinase inhibited the formation of nuclear aggresomes in a process that likely involved PML domains. A subsequent analysis of proteins associated with PML domains identified specific changes in Rb in the presence of kinase activity. Further analysis identified two Rb consensus binding sites in the amino acid sequence of pUL97 and purified Rb binds specifically to the kinase. The kinase might affect Rb directly through the consensus binding domains resulting in an alteration of PML complexes and a failure to form nuclear aggresomes. Each of Rb binding motifs was disrupted with a point mutation to assess their impact on aggresome formation. Each of these plasmids was cotransfected with pp65-EGFP to evaluate their ability to disrupt the formation of nuclear aggresomes induced by this protein. Cotransfection with a plasmid expressing the wt UL97 diminished the formation of nuclear aggresomes, while those expressing the K355M mutant or the UL27 open reading frames had no affect (Fig. 6). Disruption of the LxCxD (SEQ ID NO:2) motif (C428G) did not appear to impair the ability of the kinase to disrupt aggregates to a significant degree. However, a mutation in the LxCxE (SEQ ID NO:1) motif (Cl 5 IG) eliminated its ability of the Attorney Docket No. 20674-066WO1

kinase to inhibit aggresome formation and results from this plasmid resembled those obtained with the K355M mutant. These results indicate that the kinase affects Rb function directly through the Rb binding motifs. They are also consistent with the kinase affecting nuclear aggresome formation by a mechanism that involves Rb, mediated by PML domains. The essential lysine and the active site (K355M) and the consensus Rb binding motif LxCxE (SEQ ID NO:1) (C 15 IG) were required for the enzyme to inhibit the formation of nuclear aggresomes. Example 2. UL97 and U69 Protein Kinases Inhibit Aggregate Formation.

A surrogate assay was developed for activity of the U69 protein kinase based on its kinase dependent inhibition of nuclear aggregation. This assay is similar to that reported previously for UL97 (Prichard, M. N., et al., 2005. Human cytomegalovirus UL97 Kinase is required for the normal intranuclear distribution of pp65 and virion morphogenesis. J Virol 79:15494-502). As shown in Figure 8, aggregation of pp65- GFP was inhibited by both the HCMV UL97 kinase and the HHV-6 U69 kinase (light gray bars). Inhibition of aggregation was kinase dependent since the U69B K219M mutation, which eliminates an essential lysine, is incapable of inhibiting aggregation. Treatment of infected cells with 15 μM MBV reduced the ability of UL97 and U69 to inhibit aggregation (Figure 8, dark gray bars). These results showed that HHV-6B U69 protein kinase and UL97 were able to inhibit aggregate formation. Example 3. The UL97 Kinase Prevents Aggregation of an Aggroprobe and

PolyQ-expanded Proteins Associated with Huntington's Disease (HD) and Spinocerebellar Ataxia (SCA) 3.

GFP 170* chimeric proteins have been characterized as efficient aggroprobes (Garcia-Mata et al., J, Cell Biol. 146(6): 1239-54 (1999); Fu et al., Neurobiol Dis. 20(3):656-65 (2005)). GFP170* is a fusion of the enhanced green fluorescent protein

(EGFP) fused to the N-terminus of amino acids 566-1375 of golgin-160 (also called GCP 170). Golgin-160 is a cytoplasmic protein that associates peripherally with the cytoplasmic aspect of membranes of the Golgi complex. Under certain circumstances, golgin-160 can be detected in the nucleus, and golgin-160 contains a nuclear localization signal (NLS). Golgin-160 facilitates the trafficking of cargo proteins through the Golgi. Golgin-160 is a soluble protein with a high preponderance of coiled-coil regions and readily aggregates in cells when expressed at high levels, Attorney Docket No. 20674-066WO1

presumably due to self-aggregation of the coiled-coil regions. Golgi-160 and GFP170* do not contain polyQ repeats. When GFPO170* was transiently expressed in COS-7 or HeLa cells, it forms large aggregates in the cytoplasm and in the nucleus (Figure IA). To determine if UL97 prevents aggregation of GFP 170*, cells were co- transfected with GFP 170* and wild-type UL97. Co-transfected cells did not deposit large aggregates and instead contained a diffuse pattern of cytoplasmic and nuclear GFP 170* (Figure IB). The dispersion of GFP 170* required UL97 kinase activity, since co-expression of GFP170* with the inactive UL97/K355M did not prevent the deposition of cytoplasmic and nuclear aggregates (Figure 1C). The inactive

UL97/K355M preferentially associated with the cytoplasmic aggregates.

The cytoplasmic and nuclear aggregates formed by GFP 170* were morphologically similar to aggregates seen in cellular and animal models of HD and SCAs and in brains of patients afflicted with those diseases. Therefore, it was determined whether UL97 prevents aggregation of polyQ-expanded proteins linked to

HD and SCA3. A huntingtin-derived Httexl-82Q-YFP plasmid was used that encodes a fusion protein containing the YFP tag fused to the N-terminus of huntingtin domain corresponding to exon 1 of the protein (encoding amino acids 1-63) and containing 82 glutamine residues (Chun et al., 2001). Httexl-82Q-YFP deposited as characteristic cytoplasmic and nuclear aggregates (Figure 2A). Co-expression of wild-type UL97 with Httexl-82Q-YFP abrogated the deposition of large aggregates and resulted in a diffuse pattern of cytoplasmic and nuclear Httexl-82Q-YFP (Figure 2B). The level of dispersion varied, but, in most cells expressing the kinase, the green fluorescence was completely diffuse. The UL97 kinase was also detected within a diffuse pattern within the nucleus. The dispersion of Httexl -82Q- YFP required UL97 kinase activity, since co-expression of the inactive UL97/K355M mutant did not prevent deposition of cytoplasmic and nuclear aggregates (Figure 2C). The inactive UL97/K355M associated with the cytoplasmic and the nuclear aggregates.

The finding that UL97 inhibited Httexl -82Q- YFP agggregation showed that this viral kinase prevents aggregation of polyQ-expanded proteins implicated in neurodegenerative diseases. Attorney Docket No. 20674-066WO1

To determine if UL97 prevents aggregation of other polyQ proteins, the action of UL97 was assayed on a polyQ substrate derived from ataxin-3, the protein mutated in SCA3. AT3-72Q was used, which encodes a fusion protein containing the full length ataxin-3 with a 72-glutamine expansion fused to the amino-terminus of myc. AT3-72Q deposited as characteristic aggregates in the cytoplasm and the nucleus

(Figure 3A). Co-expression of wild-type UL97 with AT3-72Q abrogated the deposition of large aggregates and resulted in a diffuse pattern of cytoplasmic and nuclear AT3-72Q (Figure 3B). The UL97 kinase was also detected within a diffuse pattern within the nucleus. The dispersion of AT3-72Q required UL97 kinase activity since co-expression of the inactive UL97/K355M did not prevent deposition of aggregates (Figure 3C). The mutant kinase appeared to preferentially associate with the cytoplasmic, but not the nuclear aggregates of AT3-72Q.

The UL97 effect on AT3-72Q aggregation was quantitated. As shown in Figure 4A, the majority (-80%) of cells expressing AT3-72Q in the absence of UL97 contained large aggregates. Ln contrast, the majority (~65%) of cells expressing AT3-

72Q and UL97 did not contain visible aggregates and contained AT3-72Q in a diffuse pattern. The kinase activity was required for anti-aggregation, since the majority (-70%) of cells expressing AT3-72Q and the inactive UL97/K355M contained large aggregates. These results showed the efficacy of the viral UL97 kinase in preventing aggregation of non-polyQ GFP170* and polyQ Httexl-82Q and AT3-72Q proteins. The anti-aggregation function of UL97 required the kinase activity of UL97. Since preventing protein aggregation correlates with alleviation of neuronal cytotoxicity in cellular and organismal models of polyQ diseases, UL97 should reverse polyQ- induced cytotoxicity.

Claims

Attorney Docket No. 20674-066 WOlWHAT IS CLAIMED IS:

1. A method of treating or preventing a protein aggregate disease or disorder in a subject comprising administering a UL97, a UL97 homolog or a fragment or variant of a UL97 or a UL97 homolog to the subject.

2. The method of claim 1 , wherein a UL97 or fragment or variant thereof is administered to the subject.

3. The method of claim 1, wherein a UL97 homolog or fragment or variant thereof is administered to the subject.

4. The method of claim 3, wherein the UL97 homolog is EBV EGFBL4, HHV- 6AU69, HHV-6B U69 or HHV-7 U69.

5. The method of claim 1, wherein the UL97, UL97 homolog or fragment or variant of the UL97 or UL97 homolog binds Rb.

6. The method of claim 1, wherein the UL97, UL97 homolog or fragment or variant of the UL97 or UL97 homolog hyperphosphorylates Rb.

7. The method of claim 1, wherein the UL97, UL97 homolog or fragment or variant of the UL97 or UL97 homolog binds pi 30 or pi 07.

8. The method of claim 1, wherein the UL97, UL97 homolog or fragment or variant of the UL97 or UL97 homolog comprises an LxCxE (SEQ ID NO: 1) motif.

9. The method of claim 8, wherein the UL97, UL97 homolog or fragment or variant of the UL97 or UL97 homolog further comprises a DSSE (SEQ ID NO: 13) motif.

10. The method of claim 8 or 9, wherein the UL97, UL97 homolog or fragment or variant of the UL97 or UL97 homolog further comprises a LxCxD (SEQ ID NO:2) motif.

11. The method of claim 1, wherein the fragment is LxCxE (SEQ ID NO:1).

12. The method of claims 1, wherein the UL97, UL97 homolog or fragment or variant of the UL97 or UL97 homolog is administered by a dense body or tegument aggregate.

13. The method of claim 1, wherein the UL97, UL97 homolog or fragment or variant of the UL97 or UL97 homolog is administered by a virus like particle. Attorney Docket No. 20674-066WO1

14. The method of claims 1 , wherein the UL97, UL97 homolog or fragment or variant of the UL97 or UL97 homolog is administered by an expression vector.

15. The method of claim 14, wherein the expression vector is a viral vector, a bacterial vector or a plasmid.

16. The method of claim 1, wherein the protein aggregate disorder is selected from the group consisting of a disease characterized by amyloidosis, lysosomal storage disease, sickle cell disease, a prion disease, a polyglutamine repeat disease, a disease characterized by α-synuclein aggregation, a disease characterized by SODl aggregation and a protein aggregated myopathy.

17. The method of claim 16, wherein the protein aggregate myopathy is a desmin- related myopathy, an inclusion body myopathy, an actinopathy or a myosinopathy.

18. The method of claim 1, wherein the protein aggregated disorder is a neurodegenerative disorder.

19. The method of claim 18, wherein the neurodegenerative disorder is selected from the group consisting of a prion disease, Alzheimer's disease, Pick's disease, progressive supranuclear palsy, frontotemporal dementia, corticobasal degeneration, postencephalitic parkinsonism, Parkinson's disease, multiple system atrophy, Huntington's disease, Batten disease, dementia with Lewy bodies, Hallervorden-Spatz syndrome and amyotrophic lateral sclerosis disease.

20. The method of claim 16, wherein the prion disease is selected from the group consisting of Creutzfeldt- Jakob disease, bovine spongiform encephalopathy, a spongiform encephalopathy, kuru, scrapie, chronic wasting disease, fatal familial insomnia, Alper's syndrome, Grestmann-Straussler-Scheinker syndrome, transmissible mink encephalopathy, feline spongiform encephalopathy and exotic ungulate encephalopathy.

21. The method of claim 16, wherein the disorder characterized by amyloidosis is selected from the group consisting of systemic amyloidosis, familial or hereditary amyloidosis, organ-specific amyloidosis, AL amyloidosis, AA amyloidosis, gelsolin amyloidosis, Appalachian type amyloidosis, Shar Pei fever, diabetes mellitus type 2, cardiac amyloidosis, inclusion body myositis, congophilic angiopathy and Down's syndrome. Attorney Docket No. 20674-066 WOl

22. The method of claim 16, wherein the disease characterized by amyloidosis is associated with a mutation in a gene selected from the group consisting of apolipoprotein Al, lysozyme, transthyretin, apolipoprotein B and fibrinogen.

23. The method of claim 16, wherein the polyglutamine repeat disorder is selected from the group consisting of dystrophia myotonica, dentatorubro-pallidoluysian atrophy (DRPLA), Friedreich's ataxia, fragile X syndrome, fragile XE mental retardation, Machado-Joseph disease, spinobulbar muscular atrophy, spinocerebellar ataxia and Huntington's disease.

24. The method of claim 16, wherein the lysosomal storage disease is selected from the group consisting of lipid storage diseases, leukodystrophies, mucopolysaccharidoses, glycoprotein storage diseases and mucolipidoses.

25. The method of claim 1 , further comprising administering a therapeutic agent to the subject.

26. The method of claim 25, wherein the therapeutic agent is selected from the group consisting of chemotherapeutic agents, anti-inflammatory agents, anti-viral agents, anti-retroviral agents, anti-opportunistic agents, antibiotics, anticonvulsants, immunosuppressive agents, apoptosis-inducing agents, lazaroids, bioenergetics, antipsychotics, N-methyl D-aspartate (NMDA) antagonists, dopamine antagonists, antidepressants, acetylcholinesterase inhibitors, cholinesterase inhibitors, antiglutamatergic agents, dopamine receptors, dopamine agonists, immunoglobulins and pain medications.

27. The method of claims 25, wherein the therapeutic agent is selected from the group consisting of levodopa, carbidopa, benserazide, gingko biloba, qigong tolcapone, entacapone, bromocriptine, pergolide, pramipexole, ropinirole , cabergoline, apomorphine, lisuride, selegiline, rasafiline, quetiapine, rivastagime, tramiprosate, xaliproden, R-flurbiprofen and leuprolide.